Naturally occurring epidermal growth factors are polypeptides, the amino acid sequences of which for a number of vertebrate species have been reported. These include murine (Savage et al., 1972, J. Biol. Chem. 247: 7612–7621; Gray et al., 1983, Nature 303: 722–725), human (Bell et al. 1986, Nuc. Acids Res. 14: 8427–8445), rattus (Simpson et al., 1985, Eur. J. Biochem. 153: 629–637; Saggi et al., 1992, DNA and Cell Biol. 11: 481–487), porcine (Pascall et al., 1991, J. Mol. End. 6: 63–70), feline (Ohashi et al., 2002, direct submission to Genbank—accession number GI:13537341), canine (Ohashi et al., 2002, direct submission to Genbank—accession number GI:14009441), equine (PCT patent application, WO 92/16626; Stewart et al., 1994, J. Mol. End. 12: 341–350). In general, epidermal growth factors isolated from different species display a high degree of amino acid sequence identity (Carpenter and Cohen, 1979, Ann. Rev. Biochem 48:193–216; Saggi et al., 1992, DNA and Cell Biol. 11: 481–487). Analogs of epidermal growth factors are also known. (see for example, Burgess et al., 1988, Biochem 27: 4977–4985; Dudgeon et al., 1990, FEBS 261: 392–396; Saggi et al., 1992, DNA and Cell Biol. 11: 481–487; Taggart et al., 1993, Biochem. Soc. Trans. 22: 21S; U.S. Pat. No. 5,070,188). These analogs typically relate to the insertion, addition or deletion of nucleotides of the epidermal growth factor gene thereby creating a protein different from the naturally occurring epidermal growth factor.
The preparation of epidermal growth factors is well known in the art. Epidermal growth factor was initially isolated from male mouse submaxillary gland (Cohen, 1962, J. Biol. Chem 237: 1555–62.) and human urine at a concentration of 0.001 mg/L (Smith et al, 1982, Nuc. Acids. Res. 15: 4497–4482) but has also been isolated from saliva, tears, milk and blood plasma (Bennett and Schultz, 1993 Am J Surg 165:728–37; Carpenter and Cohen, 1979, Ann. Rev. Biochem 48:193–216). Epidermal growth factors can also be prepared by production in genetically engineered microorganisms, such as Escherichia coli containing recombinant DNA which encodes an epidermal growth factor polypeptide (e.g. Smith et al., 1982, Nucl. Acids Res. 10: 4467–4482; Oka et al., 1985, Proc Natl. Acad. Sci. 82: 7212–7216; U.S. Pat. No. 5,652,120; WO 94/25592; Tong et al. 2001, App. Micro. Biotech. 57: 674–679; EP 0 234 888 B1, U.S. Pat. No. 5,004,686, U.S. Pat. No. 4,743,679). Other microbial hosts like Bacillus brevis (Yamagata et al. 1989, Proc. Natl. Acad. Sci. 86: 3589–3593) and eukaryotic hosts like yeasts (see for example, Urdea et al. 1983, Proc. Natl, Acad. Sci. 80: 7461–7465; Clare et al., 1991, Gene 15: 205–212) have been used for the production of epidermal growth factor.
The low costs associated with growing plants, make plants an attractive host for the production of epidermal growth factors. To the best of the present inventors knowledge only limited success has been reported for the production of an epidermal growth factor in plants. Higo et al. 1993 (Biosci. Biotech. Biochem. 57: 1477–1481) report the expression of human epidermal growth factor in the leaves of tobacco at a level of 0.001% (approximately 60 pg/mg protein) of total soluble protein. Note that the epidermal growth factor construct was optimized for E. coli codon usage. An expression level of approximately 120 pg of epidermal growth factor per mg of total soluble proteins in potato tubers was achieved by Salmanian et al. 1996, Biotech. Lett. 18: 1095–1098. Kobayaski et al., 1996, J. Japan Soc. Hort. Sci. 64(4): 763–769 disclose the expression of 65 pg of epidermal growth factor per mg of soluble protein in the leaves of kiwi fruit and 113 pg of epidermal growth factor per mg of soluble protein in the trifoliate orange leaves. Hooker et al. disclose (WO 98/21348) an epidermal growth factor expression level of 4100 pg/mg of total soluble protein in transgenic calli. Finally Du et al., reported at the Second International Molecular Farming Conference, London, Ontario, Canada (1999) a porcine epidermal growth factor expression level of 0.12% of total protein in tobacco leaves. A review of the prior art reveals no successful accumulation of epidermal growth factor in seeds.
Although methods for producing epidermal growth factor are well known to skilled artisans, the existing methods are relatively expensive, especially when large production volumes are required. Accordingly there is a need in the art for additional economical production methods of epidermal growth factor.